G-quadruplex binding assays and compounds therefor

Abstract

The present invention provides methods for assaying binding of compounds to G-quadruplex structures. Also provided are methods for screening candidate compounds for use as modulators of G-quadruplex activity, and methods for screening candidate compounds for telomerase inhibitory activity. The invention further provides novel compounds useful in the assays of the invention.

1. A G-quadruplex analog polynucleotide comprising the structure:
   (5′-(L 1 ) q (L 2 ) r (L 3 ) s (L 4 ) t (L 5 ) u (L 6 ) v ) x    
 
       wherein:
 each L 1 , L 2 , L 3 , L 4 , L 5  and L 6  is independently selected from the group consisting of a nucleotide, a nucleotide analog, and a nucleotide comprising a 6-methyl-isoxanthopterin nucleobase; 
 q, r, s, t, u and v are each independently 0, 1 or 2, provided that the sum of q+r+s+t+u+v is at least 3; and 
 x is 1 to 8; 
 
       provided that:
 5′-(L 1 ) q (L 2 ) r (L 3 ) s (L 4 ) t (L 5 ) u (L 6 ) v is a analog of a telomeric repeat sequence; 
 said polynucleotide contains at least one nucleotide comprising a 6-methyl-isoxanthopterin nucleobase; 
 said G-quadruplex analog polynucleotide forms a G-quadruplex structure; and 
 wherein each 6-methyl-isoxanthopterin nucleobase is located within said G-quadruplex structure. 
 
     
     
       2. The G-quadruplex analog polynucleotide of  claim 1  wherein the G-quadruplex analog polynucleotide is an analog of a human telomeric repeat sequence. 
     
     
       3. The G-quadruplex analog polynucleotide of  claim 1  wherein q, r, s, t, u and v are each 1. 
     
     
       4. The G-quadruplex analog polynucleotide of  claim 3  wherein x is from 2 to 6. 
     
     
       5. The G-quadruplex analog polynucleotide of  claim 3  wherein x is from 3 to 5. 
     
     
       6. The G-quadruplex analog polynucleotide of  claim 3  wherein x is 4. 
     
     
       7. The G-quadruplex analog polynucleotide of  claim 6  wherein said G-quadruplex analog polynucleotide contains one nucleotide comprising a 6-methyl-isoxanthopterin nucleobase. 
     
     
       8. The G-quadruplex analog polynucleotide of  claim 6  wherein said G-quadruplex analog polynucleotide contains two nucleotides comprising a 6-methyl-isoxanthopterin nucleobase. 
     
     
       9. The G-quadruplex analog polynucleotide of  claim 6  wherein said G-quadruplex analog polynucleotide contains three nucleotides comprising a 6-methyl-isoxanthopterin nucleobase. 
     
     
       10. The G-quadruplex analog polynucleotide of  claim 6  wherein said G-quadruplex analog polynucleotide contains four nucleotides comprising a 6-methyl-isoxanthopterin nucleobase. 
     
     
       11. The G-quadruplex analog polynucleotide of  claim 6  wherein said G-quadruplex analog polynucleotide contains five nucleotides comprising a 6-methyl-isoxanthopterin nucleobase. 
     
     
       12. The G-quadruplex analog polynucleotide of  claim 6  wherein said G-quadruplex analog polynucleotide contains six nucleotides comprising a 6-methyl-isoxanthopterin nucleobase. 
     
     
       13. A compound comprising of any of SEQ ID NOs: 1-8wherein at least one G nucleobase therein has been replaced with a 6-methyl-isoxanthopterin nucleobase, provided that said compound forms a G-quadruplex structure; and wherein each 6-methyl-isoxanthopterin nucleobase is located within said G-quadruplex structure. 
     
     
       14. The compound of  claim 13  wherein the compound comprises any of SEQ ID NOs: 2-8. 
     
     
       15. The compound of  claim 13  wherein the compound comprises any of SEQ ID NOs: 3-5. 
     
     
       16. The compound of  claim 13  wherein the compound comprises SEQ ID NO: 4. 
     
     
       17. An array comprising the G-quadruplex analog polynucleotide of  claim 1 . 
     
     
       18. An array comprising the G-quadruplex analog polynucleotide of  claim 2 . 
     
     
       19. An array comprising the G-quadruplex analog poloynucleotide of  claim 3 . 
     
     
       20. A G-quadruplex analog polynucleotide comprising:
 a nucleobase sequence that is an analog of a telomeric repeat sequence; and 
 at least one nucleotide comprising a 6-methyl-isoxanthopterin nucleobase; 
 wherein: 
 said G-quadruplex analog polynucleotide forms a G-quadruplex; and 
 each 6-methyl-isoxanthopterin nucleobase is located within said G-quadruplex structure.